4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitization testing

Principles of method if other than guideline:

​Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 4 years experience with both the KeratinoSens and DPRA assays.

GLP compliance:

no

Remarks:

Conducted within the "spirit" of GLP (See Principles of method if other than guideline)

Type of study:

other: Direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

EC Number:

252-961-2

EC Name:

4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Cas Number:

36306-87-3

Molecular formula:

C14H24O2

IUPAC Name:

4-(1-ethoxyethenyl)-3,3,5,5-tetramethylcyclohexan-1-one

impurity 1

Reference substance name:

(3E,6E)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

Molecular formula:

C14H24O2

IUPAC Name:

(3E,6E)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

impurity 2

Reference substance name:

(E)-6-ethoxy-4,6,8-trimethylnona-3,7-dien-2-one

IUPAC Name:

(E)-6-ethoxy-4,6,8-trimethylnona-3,7-dien-2-one

impurity 3

Reference substance name:

(3E,6Z)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

IUPAC Name:

(3E,6Z)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one

impurity 4

Reference substance name:

1,3-diethoxy-5,7,7-trimethylbicyclo[3.3.1]non-2-ene

IUPAC Name:

1,3-diethoxy-5,7,7-trimethylbicyclo[3.3.1]non-2-ene

impurity 5

Reference substance name:

3,5-diethoxy-1,7,7-trimethylbicyclo[3.3.1]non-2-ene

IUPAC Name:

3,5-diethoxy-1,7,7-trimethylbicyclo[3.3.1]non-2-ene

Test material form:

liquid

Specific details on test material used for the study:

Trivial name : KEPHALIS
Chemical name : Cyclohexanone, 4-(1-ethoxyethenyl)-3,3,5,5-tetramethyl-
Molecular weight : 224.3
Molecular formula : C14H24O2
Purity : 80.9 % (sum of two main peaks)
Supplier : Givaudan Schweiz AG
Product code : 6378003
CAS number : 36306-87-3
EC number : 252-961-2
Expiration date : 19 Oct 2016
Batch number : PE00141817
Physical form : Liquid
Storage conditions : 4°C

In chemico test system

Details on the study design:

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

Experimental
The test substance KEPHALIS was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by KEPHALIS was determined by HPLC-UV.

Test System(s):
The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.

Endpoint & Endpoint Detection:
24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

Endpoint Value:
The endpoint is expressed as % peptide depletion.

Positive control
In each test Cinnamic aldehyde is included as positive control.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP.

Prediction Model
Based on the average peptide depletion values for both the Cysteine and the Lysine peptide, a reactivity class is attributed to the substances. Average depletion below 6.38% indicates minimal reactivity, substances in this class are predicted as non-sensitizers by the DPRA.
The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in Test report RCR 153’453, ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility’.

Results and discussion

In vitro / in chemico

Results

Results 1

Parameter:

other: Cys-peptide depletion

Value:

2.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Parameter:

other: Lys-peptide depletion

Value:

4.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Parameter:

other: Average depletion Cys-and Lys-peptide

Value:

3.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 4

Parameter:

other: Elution time Cys peptide

Value:

10.64

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Parameter:

other: Elution time test substance Cys peptide run

Value:

15.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 6

Parameter:

other: Elution time Lys peptide

Value:

7.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 7

Parameter:

other: Elution time test substance Lys peptide run

Value:

16.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Test substance overall results:

	Average	Standard deviation
Cys-peptide depletion	2.9	2.6
Lys-peptide depletion	4.1	0.1
Average depletion Cys-and Lys-peptide	3.5
Reactivity Class	MINIMAL
Prediction	Non-sensitizer
Elution time Cys peptide	10.64
Elution time test substance Cys peptide run	15.5 / 16.1
Elution time Lys peptide	7.97
Elution time test substance Lys peptide run	16.1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

KEPHALIS was non-reactive and classified into the MINIMAL reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Executive summary:

The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard [3-5] particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results. KEPHALIS was non-reactive and classified into the MINIMAL reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.